The development from a single cell to a fully organized organism is a complex process wherein cell division and differentiation are involved. Certain proteins play a central role in this process. These proteins are divided into different families of which the transforming growth factor β (“TGF-β”) family of ligands, their serine/threonine kinase (“STK”) receptors and their signalling components are undoubtedly key regulatory polypeptides. Members of the TGF-b superfamily have been documented to play crucial roles in early developmental events such as mesoderm formation and gastrulation, but also at later stages in processes such as neurogenesis, organogenesis, apoptosis and establishment of left-right asymmetry. In addition, TGF-b ligands and components of their signal transduction pathway have been identified as putative tumor suppressors in the adult organism.
Recently, “SMAD proteins” have been identified as downstream targets of the STK receptors (Massagué, 1996, Cell, 85, p. 947-950). These SMAD proteins are signal transducers which become phosphorylated by activated type I receptors and thereupon accumulate in the nucleus where they may be involved in transcriptional activation. SMAD proteins comprise a family of at least 5 subgroups which show high cross-species homology. They are generally proteins of about 450 amino acids (50-60 kDa) with highly conserved N-terminal and C-terminal domains, linked by a variable, proline-rich, middle region. On the basis of experiments carried out in cell lines or in Xenopus embryos, it has been suggested that the subgroups define distinct signalling pathways: SMAD1 mediates BMP2/4 pathways, while SMAD2 and SMAD3 act in TGF-b/activin signal transduction cascades. It has been demonstrated that these SMADs act in a complex with SMAD4 (dpc-4) to elicit certain activin, bone morphogenetic protein (BMP) or TGF-b responses (Lagna et al., 1996, Nature, 383, p.832-836 and Zhang et al., 1996, Nature, 383, p.168-172).
SMAD proteins have a three-domain structure and their highly conserved carboxyl domain (C-domain) is necessary and sufficient for SMAD function in the nucleus. The concept that this domain of SMAD proteins might interact with transcription factors in order to regulate transcription of target genes has previously been put forth (Meersseman et al., 1997, Mech. Dev., 61, p.127-140). This hypothesis has been supported by the recent identification of a new winged-helix transcription factor (“FAST1”) which forms an activin-dependent complex with SMAD2 and binds to an activin-responsive element in the Mix-2 promoter (Chen et al., Nature 383, p. 691-696, 1996). However, cofactors for SMAD proteins other than FAST 1 have not yet been identified.
Beyond the determination of the mechanism of activation of STK receptors and SMAD, and the heteromerization of the latter, little is known about other downstream components in the signal transduction machinery. Thus, understanding how cells respond to TGF-b related ligands remains a crucial central question in this field.
In order to clearly demonstrate that SMAD proteins might have a function in transcriptional regulation—either directly or indirectly—it is necessary to identify putative cofactors of SMAD proteins and response elements in target genes for these SMAD proteins and/or cofactors, and to investigate the ligand-dependency of these activities.
To understand those interactions, molecular and developmental biology research on (i) functional aspects of the ligands, receptors and signaling components (in particular, members of the SMAD family) in embryogenesis and disease, (ii) structure-function analysis of the ligands and the receptors, (iii) the elucidation of signal transduction, (iv) the identification of cofactors for SMAD (related) proteins, and (v) ligand-responsive genes in cultured cell and the Drosophila, amphibian, fish and murine embryo are all of utmost importance.